Cloning and expression in Escherichia coli of the 135-kDa insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7.

Shimizu, Masatoshi; Oshie, Kazuyuki; Nakamura, Keiko; Takada, Yoji; Oeda, Kenji; Ohkawa, Hideo

doi:10.1271/bbb1961.52.1565

Cited by 17 publications

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“…aizawai IPL7 exhibits insecticidal activity toward lepidopteran larvae and synthesizes mainly 130-and 135-kilodalton (kDa) insecticidal proteins (IPs) which form bipyramidal crystals. The two IP-encoding genes were cloned, and the primary structures were deduced from their nucleotide sequences (14,15 Analysis of amorphous and crystallike bodies formed in E. coli by SDS-PAGE. After E. coli JM103(pTB1) cells were cultured at 37 and 30°C, both amorphous and crystallike bodies were separately prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 11).…”

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Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli

et al. 1989

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Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130-and 135-kilodalton insecticidal proteins, respectively, of BaciUus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37°C, but bipyramidal crystals practically free of contaminants were observed at 30°C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.Bacillus thuringiensis produces proteinaceous crystals toxic to insect larvae (5,7,10). Depending on the subspecies, the crystals are specifically toxic to lepidopteran (8, 9), dipteran (12,16,17), or coleopterous (2, 4, 6) larvae. B. thuringiensis subsp. aizawai IPL7 exhibits insecticidal activity toward lepidopteran larvae and synthesizes mainly 130-and 135-kilodalton (kDa) insecticidal proteins (IPs) which form bipyramidal crystals. The two IP-encoding genes were cloned, and the primary structures were deduced from their nucleotide sequences (14,15 37°C in L broth medium (13) were examined for inclusion bodies by phase-contrast microscopy (Fig. 1A). Distinct inclusion bodies were first observed after 8 h of incubation. These continued to grow until the late exponential growth phase and finally reached a length exceeding 0.5 ,um (Fig. 1A). At the stationary growth phase, over 95% of the E. coli cells contained the inclusion bodies. No phase-refractile inclusions were found in the negative control cells of E. coli JM103(pKK223-4) (data not shown). The inclusion bodies were easily recovered from the cells by a combination of freeze-thawing, sonication, and centrifugation (4,500 x g, 5 min). Under microscopic observation, the size and shape of the inclusion bodies were not uniform (Fig. 1B). When E. coli JM103(pTB1) cells were cultured at 30°C, crystallike inclusion bodies were formed (Fig. 1C) and most were of much the same size (Fig. 1D) Analysis of amorphous and crystallike bodies formed in E. coli by SDS-PAGE. After E. coli JM103(pTB1) cells were cultured at 37 and 30°C, both amorphous and crystallike bodies were separately prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 11). The main protein band in the amorphous bodies was the 130-kDa IP, although other protein bands with lower molecular masses were found ( Fig. 2A). In Western blot (immunoblot) experiments, degraded IP bands were not observed with the cell extracts of E. coli JM103(pTB1) (14).Several washes of the inclusion bodies with 0.5 M NaCl gave similar results, indicating that the lower-molecular-mass proteins were involved in the inclusion bodies. ADC-18 densitometer (Gelman Sciences, Inc.) scanning of the gel indicated that the purity of the 130-kDa IP was about 70%. The crystallike bodies contained lesser amounts of other small p...

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Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli

et al. 1989

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“…IPs of B. thuringiensis subsp. aizawai IPL7 were prepared as described previously (32). Proteins were resolved by sodium dodecyl sulfate (SDS)-PAGE (20) and then transferred electrophoretically to nitrocellulose sheets.…”

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confidence: 99%

“…A number of lepidopteran toxin genes were cloned from several serotypes of B. thuringiensis (11), and some of them were expressed in Escherichia coli (1,7,10,18,19,28,31,32,35,36,38), B. subtilis (5, 9, 11, 18, 33), B. megaterium (34), and Pseudomonas fluorescens (26). When the IP genes were fully expressed in E. coli (27) or B. subtilis (33), well-formed crystals similar to those of the parental B. thuringiensis strains were observed in these cells.…”

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Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile

Nakamura

Imanaka

1989

Appl Environ Microbiol

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Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [kDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuyingiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus a-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis M1113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10 molecules per cell) at 37rC. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (102 to 103 molecules per cell) at 55°C.

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Expression ofcry3Agene and its toxicity against Asian Gray WeevilMyllocerus undecimpustulatus undatusMarshall (Coleoptera: Curculionidae)

Swamy

Asokan

Thimmegowda

et al. 2013

J. Basic Microbiol.

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Coleopterans are the most damaging pests of many agricultural and forestry crops; there is an urgent need to develop effective biopesticides against these insects. Enhancers of Bt toxicity typify an opportunity to improve currently available commercial products into more effective control agents against diverse pests. A 1.9 kb DNA fragment, PCR amplified from native isolates of Bt using cry3A gene specific primers was cloned in expression vector pQE-80L and then used for transformation of Escherichia coli M15 cells. The sequence of the cloned crystal protein gene showed almost complete homology with a Coleopteran active Cry3A toxin gene with 117 mutations scattered in different domain regions encoding a protein of 645 amino acid residues in length, with a predicted molecular mass of 77.4 kDa. Phylogenetic analysis could be compulsive for new/novel Bacillus thuringiensis strains, allowing them to be grouped with related Cry proteins. The toxicity of Bt protein was determined against Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) LC50 152 ng cm(-2). Genes coding for Coleopteran active Cry3A proteins have been isolated and their efficient expression will provide the tools necessary to increase the efficacy of Cry-based biopesticide against economically important beetles.

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Cloning and expression in Escherichia coli of the 135-kDa insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7.

Cited by 17 publications

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Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli

Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile

Expression ofcry3Agene and its toxicity against Asian Gray WeevilMyllocerus undecimpustulatus undatusMarshall (Coleoptera: Curculionidae)

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